We have studied the post translational modifications of the insulin receptor, i.e., glycosylation, fatty acylation, and phosphorylation using biosynthesis labeling of the insulin receptor. Based on the differential sensitivity of tryptic peptides biosynthetically labeled with radioactive sugars to endopeptidases, we have shown that the insulin receptor contains O-linked oligosaccharides. The peptide containing the O-linked oligosaccharides was localized to the amino terminal tryptic peptide of the beta subunit by specific immunoprecipitation with an anti-peptide antibody to the 12 amino acids at the amino terminus of the beta subunit. Using an inhibitor of O-linked glycosylation, benzyl-N-acetyl-galactosaminide, we confirmed the finding of O-linked oligosaccharides and investigated their function. In the presence of this inhibitor the binding of insulin to the insulin receptor and the autophosphorylation of the beta subunit was unchanged. The investigation of fatty acylation of the insulin receptor, previously reported by us in IM-9 lymphocytes, was extended to now show acylation of the receptor overexpressed in 3T3 fibroblasts transfected with the cDNA for the human insulin receptor. Both [(3)H]myristic and [(3)H]palmitic acid labeled the proreceptor and the mature subunits. The time course of labeling showed early labeling of the proreceptor and later appearance of label in the mature subunits. Thus, acylation of the insulin receptor is not limited to a single cell type.